EP0157574B1 - Hybridome humain-humain produisant un anticorps antitétanique et procédé pour sa production - Google Patents

Hybridome humain-humain produisant un anticorps antitétanique et procédé pour sa production Download PDF

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Publication number
EP0157574B1
EP0157574B1 EP85302070A EP85302070A EP0157574B1 EP 0157574 B1 EP0157574 B1 EP 0157574B1 EP 85302070 A EP85302070 A EP 85302070A EP 85302070 A EP85302070 A EP 85302070A EP 0157574 B1 EP0157574 B1 EP 0157574B1
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Prior art keywords
human
cell
cells
cell line
producing
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EP85302070A
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German (de)
English (en)
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EP0157574A3 (en
EP0157574A2 (fr
Inventor
Yasuo Amatsuji
Hideyuki Ishikawa
Hirofumi Arimura
Masayuki Nishida
Tadakazu Suyama
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Mitsubishi Tanabe Pharma Corp
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Green Cross Corp Japan
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a novel human-human hybridoma. More particularly, it relates to a human-human hybridoma having antitetanic antibody producing ability.
  • the human-human hybridoma according to this invention can be utilized for the production of a monoclonal antibody against Clostridium tetani. Further, since the cell line is of human origin, the antibody produced by said hybridoma causes no trouble due to antigenicity. Thus, a great therapeutic effect can be produced by this invention.
  • antitetanus antibody has an effect of preventing the onset of tetanus by administration to persons infected with Clostridium tetani. Further, it has been revealed recently that the administration of tetanus antibody can prevent not only the onset of tetanus but also the infection itself. Accordingly, the antibody is expected to make a great medical contribution.
  • This monoclonal antibody is a single antibody which reacts against only one kind of antigenic determinant. Preparation thereof can be conducted at present by either cell fusion method or transformation method. In both methods, there are prepared cells having concurrently both proliferative ability and antibody producing ability.
  • the former method comprises fusion of B-cells of an immunized donor with B cell line in vitro and the latter comprises infecting B-cells of an immunized donor with lymphotropic viruses such as Epstein Barr virus to transform the former cells into immortalized cells.
  • the proliferating B cell line is prepared from a human B cell line GM 1500 rendered resistant to 6-thioguanine (and therefore HAT sensitive) by a mutagenesis treatment using ethyl methane sulphonate; see Croce et ai, Nature, (Dec. 1980), 288, 488-489.
  • the HAT sensitive B cell line is rendered resistant to ouabain by a mutagenesis treatment using y-radiation (200­R). Subsequent treatment, including subculturing in gradually increasing concentrations of ouabain, yields a cell line KR4 found to be resistant to 0.5mM ouabain 5 months after mutagenesis. However, such mutagenesis treatments are considered likely to cause mutation in genes other than that of ouabain.
  • This invention provides a human-human hybridoma for antitetanic antibody production which has an excellent proliferative ability and is free from the problem of antigenicity, and provides a human-human hybridoma obtained by fusing the human, HAT sensitive, ouabain resistant B cell line with a human, antitetanic antibody producing B limphoblastoid cell transformed with a lymphotropic virus.
  • the cell fusion in this invention is thus conducted by using a cell having a proliferative ability and a cell having an antibody producing ability.
  • the antibody producing cell used in this invention is a human, antitetanic antibody producing lymphoblastoid transformed with a lymphotropic virus.
  • lymphoblastoid cell can be prepared by known methods, for example in the following manner.
  • the tetanus antigen used to obtain said lymphoblastoid cell is an exotoxin which is a protein having a molecular weight of about 150,000 and an isoelectric point of 5.1 produced by Clostridium tetani. It can usually be prepared by purifying a commercially available detoxicated toxoid or a culture fluid conditioned by Clostridium tetani. It is also possible to subject the tetanus antigen thus obtained to a heat treatment in the presence of a protein denaturing agent to enhance its antigenicity. The antigen is preferably further purified for immunization.
  • a B cell may then be stimulated with above-mentioned tetanus antigen for antibody production and this stimulation may be conducted either in vivo or in vitro.
  • Stimulation in vivo can be conducted by known methods.
  • the intended human, antitetanic antibody producing B cells can be obtained by inoculating a human being with the tetanus antigen intracutaneously 2 to 3 times collecting the blood several days after final immunization, then recovering B cells from the blood obtained above followed by purification thereof.
  • the stimulation with tetanus antigen in vitro can be conducted, for example, by bringing a suitable amount of purified antigen into contact with the B cells at 30 to 40°C for 10 to 50 hours, generally in a culture medium.
  • the separation of the antitetanic antibody producing B cells is preferably conducted by means of Ficoll-Conray density gradient centrifugation.
  • the B cell thus obtained is then transformed into the immortalized cell by using a lymphotropic virus.
  • Lymphotropic viruses such as Epstein Barr virus
  • Epstein Barr virus derived from the marmoset cell line B 95-8 (free from mycoplasmas; obtained as a supernatant from B 95-8).
  • a suitable amount of the virus thus prepared is added dropwise to a medium conditioned by B cells and the virus is contacted with the B cells at 37°C for 5 to 20 days.
  • the cultivation of B cells is conducted, for example, in 5% fetal bovine serum at 37°C.
  • the cultivation may also be conducted in a culture medium supplemented with glutamine.
  • the immortalized B cells obtained thus by transformation are subcultured and those cells which produce antibodies against tetanus antigen are selected out of the subcloned cells and concentrated.
  • the antitetanic antibody producing cells can be traced by such methods as, for example, PHA, EIA and RIA.
  • a human, HAT sensitive, ouabain resistant B cell is used in this invention as the cell having proliferative ability.
  • the said human, HAT sensitive, ouabain resistant B cell line is prepared as follows.
  • An autogenetic HAT sensitive cell is obtained by gradually increasing the concentration of 6-thioguanine in the medium, optionally over a long time, in cultivation of human B cell line.
  • the HAT sensitive, B cell line is rendered ouabain resistant by increasing the concentration of ouabain (such increase may be gradual) in the culturing medium of the HAT sensitive B cell line and collecting the cells which survive.
  • ouabain resistance such increase may be gradual
  • cell fusion is conducted by using the human, antitetanic antibody producing B cells having proliferative ability owing to transformation, that is, human antitetanic antibody producing lymphoblastoid cells, and the human, HAT sensitive, ouabain resistant B cell line obtained above.
  • the cell fusion is conducted in a manner known per se.
  • One example thereof comprises reacting immortalized cells with the aforementioned lymphoblastoid cell capable of producing antibodies in the presence of polyethylene glycol.
  • a suitable mixing ratio of the cells is 1 to 10 lymphoblastoid cells relative to one immortalized cell.
  • the fused cells thus obtained are subjected to selection using a medium supplemented with HAT and ouabain.
  • the selection can be conducted based on the following ground.
  • the HAT sensitivity means the property of a cell that it cannot grow or proliferate in the presence of HAT in the medium.
  • the ouabain resistance of a cell means that the cell can proliferate even in the presence of a high concentration of ouabain. It is also known that the fusion of an ouabain resistant cell and a normal one results in an ouabain resistant cell.
  • lymphoblastoid cells being devoid of both HAT sensitivity and ouabain resistance
  • both HAT and ouabain are present in the medium
  • neither unfused B cell line nor unfused lymphoblastoid cell can proliferate, the former because of HAT sensitivity and the latter because of not being ouabain resistant, whereas successfully fused cells can proliferate because of being ouabain resistant. It is possible thus to select the fused cells alone.
  • the antitetanic antibody producing human-human hybridoma can be obtained by further screening and cloning the cells producing the intended antitetanic antibody by a known method.
  • an antibody producing cell which is excellent in antibody producing ability and particularly in proliferative ability as compared with those obtained by simply fusing human HAT sensitive B cell line with human, antitetanic antibody producing B cells or by merely transforming human, antitetanic antibody producing lymphoblastoid cells transformed with Epstein Barr virus.
  • monoclonal antibodies By proliferating the hybridoma in a growth medium, monoclonal antibodies can be produced continuously and then recovered from the medium.
  • tetanus toxoid 0.5 ml
  • Blood was collected at the about 4th day from the injection.
  • Lymphocytes were isolated from the peripheral blood collected above by means of Ficoll-Conray density gradient centrifugation and then T cells were removed therefrom by means of E-rosette formation to obtain B cells alone.
  • the B cells were then cultivated in an RPMI 1640 supplemented with 15% fetal bovine serum and 2 mM glutamine medium (cell density: 1 x 10 6 cells/ml).
  • B 95 ⁇ 8 cells (Marmoset established lymphocyte cell) were cultivated in an RPMI 1640 supplemented with 20% fetal bovine serum medium and the supernatant culture fluid was collected after several days of cultivation to be used as Epstein Barr virus liquid (concentration: 10 5 transforming dose/ml).
  • Epstein Barr virus liquid concentration: 10 5 transforming dose/ml.
  • One ml of the above-mentioned medium conditioned by B cells was mixed with 0.2 ml of the Epstein Barr virus liquid to effect infection and then subjected to static culture for three weeks in a medium (RPMI 1640 supplemented with 20% fetal bovine serum) under 5% C0 2 atmosphere to proliferate the cells.
  • Recovery of antibody producing cells from the proliferated ones was conducted by limiting dilution culture method while checking by ELISA method.
  • subcultured cells having antitetanic antibody producing ability were obtained.
  • human, HAT sensitive, B cell line were prepared in the following manner. Human B cell line in their logarithmic growth phase were cultured with a medium (RPMI 1640 supplemented with 10% fetal bovine serum) containing 0.05 pg/ml of 6-thioguanine. Then surviving cells were cultured in gradually increasing concentrations of 6-thioguanine, and after 3 months, the cells were found to be resistant to 6-thioguanine and showed high sensitivity to HAT.
  • a medium RPMI 1640 supplemented with 10% fetal bovine serum
  • Human HAT sensitive, ouabain resistant B cell line were prepared in the following manner.
  • the above-mentioned human, HAT sensitive B cell line in their logarithmic growth phase was diluted in a medium (RPMI 1640 supplemented with 10% fetal bovine serum) to a density of 8 cells/ml.
  • the diluted liquid was dispensed onto a 96-well microplate at a rate of 100 ⁇ l/well to give a number of cells per well of 0.8.
  • the plate was incubated at 37°C under 5% C0 2 atmosphere for about 2 weeks. After the period, proliferation of cells was observed to be occurring in a cloning efficiency of 30 to 40%.
  • the most proliferative cells among these were adjusted to a cell density of about 10 6 cells/ml, the resulting culture fluid was mixed with 10- 7 M, ouabain, and cultivation was conducted in the mixed medium. Similar medium exchange was repeated 2 to 3 times and finally cultivation was conducted in a medium containing 10- 6 M of ouabain. Thereafter, cultivation was conducted in a medium containing no ouabain and a very small number of cells which had survived were proliferated. After about 10 days the proliferated cells were washed by centrifugation, suspended in a medium containing 10- 6 M of ouabain to a density of 10 5 cells/ml and the suspension was dispensed onto a 96-well microplate at a rate of 100 pl/well. The plate was incubated for about one month, the medium being exchanged 2 times a week. Thus, cells were obtained which can proliferate even in the presence of 10- 6 M ouabain.
  • the HAT sensitive, ouabain resistant B cell line (1.5 x 106 cells) and the aforementioned lymphoblastoid cells (3 x 10 6 cells) were mixed, and the mixture was further mixed with 0.3 ml of 50% polyethylene glycol (PEG 1500, mfd by BDH) and was incubated at 37°C for 2 minutes to effect cell fusion. After washed. by centrifugation, the cells were adjusted to a density of 1 x 10 6 cells/ml with an RPMI 1640 supplemented with 10% fetal bovine serum medium and resulting liquid was dispensed onto a 96-well microplate at a rate of 100 pl/well.
  • PEG 1500, mfd by BDH polyethylene glycol
  • a HAT + ouabain selective medium [final concentration: hypoxanthine 13.61 mg/l, aminopterin 0.18 mg/l, thymidine 3.88 mg/I, ouabain 0.5 x 10 -6 M] was added 24 hours after the PEG treatment and cultivation was then conducted for 2 weeks.
  • Example 1 Two strains of hybridomas (10C, 10D) obtained in Example 1 were examined for their number of chromosomes (Table 1). The values in the Table show the number of cells having the indicated number of chromosomes.
  • the hybridomas had several times (up to thirty times) more chromosomes than their parent cells, lymphoblastoid cells and HAT sensitive, ouabain resistant B cell line (each having 46 chromosomes) had.
  • the fact that said cells are hybridomas differing from the parent cells is suggested also from the number of chromosomes.
  • Hybridoma formation or transformation efficiency was indicated by the number of hybridomas or transformed cells emerging per 1 x 10 6 parent cells.
  • Crossing efficiency used as an index signifying the proliferative ability, indicates the probability of proliferatable hybridomas appearing in the whole emerging hybridomas.
  • human B cell line and human, antitetanic antibody producing B cells which are both the parent cells of the present hybridoma, as well as hybridoma between B cell line and normal B cells and lymphoblastoid cell transformed with Epstein Barr virus.
  • the established human-human hybridoma of this invention can become a very effective antitetanic antibody producing cell with a good emerging rate as well as a good proliferative ability. Moreover, the hybridoma of this invention causes no problem due to antigenicity because it is of human origin.
  • the monoclonal antibody obtained by this invention was tested for its class and specificity.
  • the sample antibody used in the test was produced by human-human hybridoma cell line used in Experimental Example 1.
  • the class of monoclonal antibody was determined (Enzyme Linked Immunosorbent Assay) using antibody against various classes of human immunoglobulin.
  • the specificity of monoclonal antibody was determined by assaying the reactivity against the fragment of tetanus toxin separated by SDS-PAGE (polyacrylamidegel) electrophoresis using the method of Western Blotting. The results are shown in Table 3.
  • the class of monoclonal antibody was gamma and kappa for 10C cell, and mu and lambda for 10D cell.
  • the specificity of monoclonal antibody produced by the 10C cell or 10D cell have recognized ⁇ fragment proposed by Helting et al or a fragment proposed by Matsuda et al, respectively.

Claims (11)

1. Procédé de préparation d'un anticorps antitétanique produisant un hybridome humain-humain, lequel procédé comprend les étapes consistant à:
a) préparer une lignée cellulaire B humaine résistant à l'ouabaïne, sensible au HAT, et
b) fusionner la lignée cellulaire B humaine résistant à l'ouabaïne et sensible au HAT avec une cellule lymphoblastoïde humaine, produisant des anticorps antitétaniques ayant été transformée par un virus lymphotrope, caractérisé en ce que l'étape a) comprend les étapes consistant à:
i) rendre une lignée cellulai re B humaine résistant à la 6-thioguanine, et par conséquent sensible au HAT, par culture de la lignée cellulaire en présence de concentrations progressivement croissantes de thioguanine, puis
ii) rendre la lignée cellulaire B humaine sensible au HAT, résistant à l'ouabaïne par culture de ladite lignée cellulaire dans des concentrations croissantes d'ouabaïne.
2. Procédé selon la revendication 1, dans lequel la cellule lymphoblastoîde humaine produisant des anticorps antitétaniques est préparée par stimulation d'une cellule lymphoblastoïde humaine avec un antigène du tétanos in vivo ou in vitro.
3. Procédé selon la revendication 2, dans lequel la stimulation in vivo est effectuée par inoculation à un être humain d'un antigène du tétanos biotransformé, et rassemblement des cellules B stimulées.
4. Procédé selon la revendication 2, dans lequel la stimulation in vitro est effectuée par mise en contact d'un antigène du tétanos biotransformé avec des cellules B humaines dans un milieu de culture, et rassemblement des cellules B stimulées.
5. Procédé selon l'une quelconque des revendications précédentes, dans lequel le virus lymphotrope est le virus d'Epstein Barr.
6. Procédé selon l'une quelconque des revendications précédentes, dans lequel la transformation a été réalisée par mise en contact de la cellule B humaine produisant des anticorps antitétaniques avec le virus lymphotrope dans un milieu de culture conditionné par ladite cellule B.
7. Procédé selon l'une quelconque des revendications précédentes, dans lequel la fusion cellulaire s'effectue par utilisation de polyéthylène glycol.
8. Procédé selon l'une quelconque des revendications précédentes, dans lequel la fusion cellulaire s'effectue selon un rapport de 1 à 10 desdites cellules lymphoblastoîdes à une cellule de ladite lignée cellulaire B résistant à l'ouabaïne et sensible au HAT.
9. Hybridome humain-humain capable de produire un anticorps monoclonal contre le Clostridium tetani, produit par le procédé de l'une quelconque des revendications 1 à 8.
10. Procédé pour la préparation d'un anticorps monoclonal actif contre le Clostridium tetani, qui comprend la culture d'un hybridome humain-humain produit par le procédé de l'une quelconque des revendications 1 à 8.
11. Anticorps monoclonal produit par le procédé de la revendication 10.
EP85302070A 1984-03-28 1985-03-26 Hybridome humain-humain produisant un anticorps antitétanique et procédé pour sa production Expired EP0157574B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP60061/84 1984-03-28
JP59060061A JPS60203186A (ja) 1984-03-28 1984-03-28 ヒト−ヒトハイブリド−マ

Publications (3)

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EP0157574A2 EP0157574A2 (fr) 1985-10-09
EP0157574A3 EP0157574A3 (en) 1986-06-18
EP0157574B1 true EP0157574B1 (fr) 1989-12-06

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EP85302070A Expired EP0157574B1 (fr) 1984-03-28 1985-03-26 Hybridome humain-humain produisant un anticorps antitétanique et procédé pour sa production

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EP (1) EP0157574B1 (fr)
JP (1) JPS60203186A (fr)
KR (1) KR850006705A (fr)
DE (1) DE3574601D1 (fr)
ES (1) ES8702791A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8426464D0 (en) * 1984-10-19 1984-11-28 Technology Licence Co Ltd Monoclonal antibodies
JP2599123B2 (ja) * 1985-12-28 1997-04-09 萩原 義秀 新規なヒトｂセル・リンパ芽球細胞変異株
US5001065A (en) * 1987-05-27 1991-03-19 Cetus Corporation Human cell line and triomas, antibodies, and transformants derived therefrom
US5126259A (en) * 1987-12-24 1992-06-30 Takeda Chemical Industries, Ltd. Human b. lymphoblastoid cell, hybridoma, antibody and production of antibody
JPH0249597A (ja) * 1988-04-27 1990-02-19 Kimiyoshi Tsuji モノクローナル抗体及びその利用方法
CA2028815A1 (fr) * 1989-03-20 1990-09-21 Yuko Mizuno Anticorps monoclonaux humains reagissant a pseudomonas aeruginosa, cellules capables de produire lesdits anticorps, methodes de production et preparations pharmaceutique
DK0562132T3 (da) * 1992-03-23 1999-03-22 Schweiz Serum & Impfinst Monoklonale antistoffer mod tetanustoksin og farmaceutiske præparater indeholdende dem
JP2721817B2 (ja) * 1995-05-15 1998-03-04 萩原 義秀 ヒト／ヒト・ハイブリドーマ及びその生産する抗体

Also Published As

Publication number Publication date
ES541574A0 (es) 1987-01-16
ES8702791A1 (es) 1987-01-16
JPS60203186A (ja) 1985-10-14
EP0157574A3 (en) 1986-06-18
DE3574601D1 (de) 1990-01-11
KR850006705A (ko) 1985-10-16
EP0157574A2 (fr) 1985-10-09

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